Multi-lab, multi-enzyme study demonstrates the versatility of bacterial microcompartment shells as a modular platform...

Area of Science:

• Biochemistry
• Synthetic Biology
• Protein Engineering

Background:

• Bacterial microcompartments (BMCs) are protein organelles that organize metabolic reactions.
• They offer a promising scaffold for engineering metabolic pathways.
• Enzyme encapsulation within BMCs can enhance stability and function.

Purpose of the Study:

• To develop and validate a modular BMC shell-based platform for in vitro enzyme encapsulation.
• To assess the generality and efficiency of the encapsulation system using various dehydrogenases.
• To evaluate the impact of encapsulation on enzyme activity, stability, and cooperative function.

Main Methods:

• Utilized the SpyCatcher-SpyTag (SC-ST) covalent conjugation system for enzyme attachment to BMC proteins.
• Expressed and purified SC-tagged dehydrogenases and ST-fused BMC-T1 proteins.
• Induced self-assembly of BMC shells (HT1) around conjugated enzymes.
• Assessed enzyme activity, kinetic parameters, thermal stability, and storage stability post-encapsulation.

Main Results:

• Successfully encapsulated 12 out of 13 expressed dehydrogenases into self-assembled BMC shells.
• Encapsulation generally retained enzymatic activity, with some modulation of kinetic parameters.
• Encapsulated enzymes exhibited enhanced thermal stability (up to ~50 °C) and storage stability.
• Demonstrated successful co-encapsulation and functional cooperation of two enzymes via cofactor recycling.
• Lyophilized and reconstituted enzyme-loaded shells retained structural integrity and activity.

Conclusions:

• Engineered BMCs provide a versatile and modular platform for organizing multi-enzyme pathways.
• The SC-ST conjugation system facilitates rapid assembly and functional integration of enzymes within BMCs.
• This approach enables enhanced enzyme stabilization and cooperative function for metabolic engineering applications.